JP7531407B2 - Microfluidic device for concentrating particles - Google Patents

Description

The present invention relates generally to techniques for detecting or estimating the amount of particles, particularly low concentrations and small amounts, present in a fluid sample.

The present invention has been developed with particular reference to microfluidic devices designed to be subjected to centrifugation, as well as devices and methods for carrying out tests or analyses of fluid samples, preferably containing organic or biological particles or bacteria or microorganisms, for example for the rapid running of antibiotics.

The present invention may be applied to the detection of other types of particles that may be present in a fluid sample, not necessarily organic or biological fluids or particles, and not necessarily via centrifugation in any case.

Various techniques are known for counting particles, e.g. cells, present in a sample of a fluid, e.g. a biological fluid. The most commonly used systems are of the optical type (with or without fluorescence), the impedance measurement type or the static type with image recognition. These known systems generally require a relatively large amount of sample and do not allow for an efficient parallelization of measurements, such as a large number of measurements performed simultaneously; that is, they assume a significant amount of starting sample to be able to perform many measurements in parallel and/or simultaneously.

Known systems based on image recognition techniques can be used to analyze small fluid samples, but do not allow the parallelization of a large number of samples, unless investments are made, resulting in long measurement times that often prove counter-economic.

In its general terms, the invention aims to indicate a device and a method that makes it possible to carry out in a simple, fast and cheap way the quantification and/or identification of particles present in low concentrations and/or in small quantities in a fluid sample, allowing parallelization in an equally simple and cheap way between a large number of samples, with advantages in terms of efficiency in terms of time and costs, and in terms of sensitivity and reproducibility.

A further object of the present invention is to show a methodology that allows to perform an antibiogram (when a microorganism is being measured), i.e. to obtain an antibiotic susceptibility profile of at least one microorganism, or pathogen, or bacterium, in a relatively short time, representative of a few hours. A subsidiary object of the present invention is to show a methodology that allows to perform multiple antibiotics simultaneously.

The above-mentioned objects are achieved according to the present invention by a microfluidic device for concentrating particles, and by a corresponding support and method presenting the characteristics specified in the appended claims. The present invention also relates to a centrifugation and/or detection device that can be used in combination with the aforementioned microfluidic device, as well as an analytical methodology based on the use of such a device.

As will become apparent below, the present invention allows for effective detection of the amount of particles in a relatively modest sample of a fluid of interest in a simple and rapid manner.

Further objects, features and advantages of the present invention will become apparent from the following detailed description, given purely by way of non-limiting example, with reference to the accompanying drawings, in which:

FIG. 1 is a schematic perspective view of a centrifugation and/or detection device and a microfluidic device according to a possible embodiment of the present invention. FIG. 1 is a schematic perspective view of a centrifugation and/or detection device and a microfluidic device according to a possible embodiment of the present invention. FIG. 1 is a schematic perspective view of a microfluidic device according to a possible embodiment of the present invention. FIG. 1 is an exploded schematic diagram of a microfluidic device according to a possible embodiment of the present invention. FIG. 5 is an enlarged detailed view of FIG. 4. FIG. 1 is a partial and schematic perspective view of a portion of a microfluidic device according to a possible embodiment of the present invention. 1 is an enlarged detail of an edge of a microfluidic device according to a possible embodiment of the present invention. 1A-1D are schematic perspective views intended to illustrate possible steps of loading a fluid sample into a microfluidic device, according to possible embodiments of the present invention. FIG. 1 is a schematic perspective, partially sectioned view of a microfluidic device according to a possible embodiment of the present invention. FIG. 10 is an enlarged detailed view of FIG. FIG. 1 is an exploded schematic diagram of a microfluidic device according to a possible embodiment of the present invention. FIG. 1 is a schematic perspective, partially sectioned view of a microfluidic device according to a possible embodiment of the present invention. FIG. 1 is a schematic perspective, partially sectioned view of a microfluidic device according to a possible embodiment of the present invention. FIG. 14 is an enlarged detailed view of FIG. 13. FIG. 2 is an enlarged detail of an end region of a microfluidic device according to a possible embodiment of the present invention. FIG. 3 is a partial cross-sectional perspective view of the device of FIGS. 1-2 with a corresponding microfluidic device in an operational state. FIG. 17 is an enlarged detailed view of FIG. 16. FIG. 1 is a schematic perspective view of a centrifugation and/or detection device and several microfluidic devices according to possible embodiments of the present invention. FIG. 19 is an enlarged detailed view of FIG. 18. FIG. 20 is a schematic perspective, partially cross-sectional view of the centrifugation device of FIG. 18 with a corresponding microfluidic device in an operational state. FIG. 1 is a schematic perspective view of a microfluidic device according to a possible embodiment of the present invention. FIG. 1 is an exploded schematic diagram of a microfluidic device according to a possible embodiment of the present invention. FIG. 1 is an exploded schematic diagram of a microfluidic device according to a possible embodiment of the present invention. FIG. 22 is a schematic perspective view intended to illustrate possible steps for loading a fluid sample into a microfluidic device of the type shown in FIG. 21 . FIG. 1 is a schematic perspective, partially cross-sectional view of a microfluidic device according to a possible embodiment of the present invention. FIG. 1 is a schematic perspective, partially cross-sectional view of a microfluidic device according to a possible embodiment of the present invention. FIG. 2 is a schematic perspective, partially sectioned view of a microfluidic device according to another possible embodiment of the present invention. 1 is a schematic perspective view intended to illustrate a possible mode of use of a microfluidic device according to a possible embodiment of the present invention; 1 is a schematic perspective view intended to illustrate a possible mode of use of a microfluidic device according to a possible embodiment of the present invention;

Reference to an "embodiment" or "one embodiment" in the framework of this specification is intended to indicate that a particular configuration, structure, or feature described in connection with the embodiment is included in at least one embodiment. Thus, phrases such as "in one embodiment," "in one embodiment," "in various embodiments," etc., which may be present at various points in this specification, do not necessarily refer to one and the same embodiment. Furthermore, particular forms, structures, or features defined in the framework of this specification may be combined in any suitable manner in one or more embodiments, even if different from those depicted. Reference numbers and spatial references (such as "above," "below," "upper," "lower," etc.) used in this specification are provided merely for convenience and therefore do not define the scope of protection or the scope of the embodiments. The same reference numbers are used in the figures to indicate elements that are similar or technically equivalent to each other.

With initial reference to Figures 1 and 2, generally indicated at 1 is a centrifugation and/or detection device having a structure 2 defining a processing and/or detection chamber 3.

In various embodiments, device 1 includes a lid or door 4, preferably hinged to structure 2, for closing chamber 3. Device 1 has a drive or movement system, generally designated 5 in FIG. 2, that includes a rotating member 5a within chamber 3 designed to rotate one or more microfluidic devices, preferably of the centrifugable type.

Possibly, the lid 4 may comprise a corresponding part 4a of a positioning and/or guiding system of a centrifugable microfluidic device or of a support configured to support a number of centrifugable microfluidic devices. In the example, part 4a comprises a seat for blocking and guiding elements, indicated by 5b, which can be coupled to member 5a, between which said centrifugable device or said support is set, to ensure a mutual fixation of rotation between the mentioned parts.

The actuation system 5 preferably comprises an electric motor (partially shown in Figs. 16-17 and designated 5c) possibly equipped with a motor reducer and/or an electronic control circuit. The centrifugation speed may preferably be indicated between 200 and 1200 rpm, preferably between 400 and 1000 rpm, for a time period comprised between 3 and 30 seconds, very preferably between 5 and 15 seconds.

In various embodiments, the device 1 comprises a system for controlling the temperature and/or humidity in the processing chamber 3. In various embodiments, this system is configured to maintain a temperature higher than 25°C, preferably between 36°C and 38°C, and/or a humidity preferably higher than 95%. In various preferred embodiments, the device 1 comprises a suction system and/or a system for regulating pressure, previously arranged to maintain the centrifugation region or chamber 3 at a pressure lower than the ambient pressure, and/or for forcing the flow of air at the output from said region or chamber to a filtration system configured to prevent the spread of potentially contaminated aerosols to the environment.

In various embodiments, the device 1 comprises a control panel, indicated by 6, such as the one only represented in Figs. 1 and 2, in which are arranged suitable control elements 6a for starting and/or stopping the centrifugation and/or regulation and/or pressure regulation and/or detection processes, and possibly for setting the parameters of said processes (e.g. centrifugation speed and/or time and/or temperature and/or humidity and/or pressure in the chamber 3), and possible indication and/or warning elements. Said control elements may be of any suitable type (push buttons, knobs, sliders, touch displays, etc.).

With reference also to FIG. 3, indicated at 10 is a microfluidic device according to a possible embodiment of the invention. In various embodiments, such as the one illustrated, the device 10 is configured to integrate or house at least one device designed to concentrate particles contained in a sample of a fluidic substance by centrifugation. To this end, the device 10 includes or integrates at least one microfluidic device, indicated by M in FIG. 3, preferably multiple microfluidic devices. In the following, for simplicity, reference will first be made to the case of a device 10 with multiple microfluidic devices M, although in other embodiments described below, the microfluidic device 10 according to the invention may include only one microfluidic device.

In various embodiments, and as illustrated in FIG. 4, the microfluidic device comprises a substrate 11 and a cover element 12 that define the or each respective portion of the microfluidic device M.

In various embodiments, it is assumed that the device 10 is set to rotate with respect to a center of rotation, identified here by the member 5a of the device 1 of Figs. 1 and 2. To this end, in various preferential embodiments, the device 10 is disk-shaped and preferentially comprises means 11a for coupling to the operating system of a corresponding centrifugation device, for example to the member 5a of the device 1 of Figs. 1 and 2. In the illustrated case, said coupling means 11a comprises a central passage or hole in a disk-shaped substrate 11. On the other hand, as can be seen, the disk shape of the substrate 11 does not constitute an essential characteristic, and this does not derogate from the fact that, in various embodiments, the substrate should be set to rotate with respect to a center of rotation.

In various embodiments, the substrate 11 is constructed with a relatively small thickness, for example between 0.5 and 4 mm. It is for example made of glass or plastic (for example polycarbonate, or polyethylene, or cycloolefin copolymer or COC) and is presented with a diameter between 10 and 30 cm, thus possibly resembling a classical compact disc. The materials used are preferentially electrically insulating materials and very preferably materials that are at least partially transparent.

In various embodiments, the cover element 12 is also constructed with a relatively thin thickness, for example between 0.1 and 0.5 mm. The cover element 12 is made, for example, from polycarbonate or COC or polyethylene or glass and has a diameter similar to that of the substrate 11, for example between 10 and 30 cm as shown.

The material or materials used for the cover element are preferentially substantially impermeable to air and liquid. Also, the cover element 12 may be disc-shaped, preferably with a central passageway 12a concentric with the passageway 11a of the substrate 11 (see, for example, FIG. 3). The cover element 12 may be made, for example, from a flexible sheet material that is bonded or adhered onto the substrate 11.

Referring to Figures 4 and 5, in various embodiments, at least one microfluidic device of a device according to the invention comprises a respective set of microchannels 13 defined in a surface 11b (here also conventionally defined as the top surface) of a substrate 11 to which a cover element 12 is applied.

In various preferred embodiments, the device 10 has a plurality of microfluidic devices that are not necessarily identical to one another. To this end, on the substrate 11, several sets of microchannels 13 may be provided, each set belonging to a respective microfluidic device. The microchannels 13 of the different sets preferably have substantially the same length, even if this does not constitute an essential characteristic.

In various embodiments, several sets of microchannels 13 of various lengths are provided. For example, in Figures 4 and 5, 13 ₁ denotes a set of microchannels of maximum length, 13 ₂ denotes a set of microchannels of minimum length and 13 ₃ denotes a set of microchannels of intermediate length. Sets of microchannels with different lengths may be useful to optimize the occupation of the space available on the substrate 11, for example, in particular with substrates having a circular shape and/or with sets of microfluidic devices or channels 13 in substantially radial positions to make available a large number of microfluidic devices on the substrate 11, and thus perform the parallelization of a large number of samples in a convenient manner.

In various embodiments, such as the one illustrated, the microchannels 13 of each set extend substantially radially with respect to the center of rotation of the device 10, i.e., with respect to the central passage 11a of the substrate 11. The microchannels 13 of each set are preferably arranged side by side, preferably parallel to one another, and/or are preferentially substantially straight. The microchannels 13 of each set extend preferentially according to a plane identified by the substrate 11, and for this purpose they can be defined on the surface 11b by suitable techniques, for example, microetching, molding, or polymerization of a resin by UV. In any case, the formation of the microchannels by deposition of a material on the substrate 11 is not excluded from the scope of the present invention.

According to the preferred embodiment shown, each set of microchannels 13 comprises at least one intermediate microchannel set that is in a radial position relative to the center of the passage 11a of the substrate 11, while the other microchannels of the same set are parallel to said intermediate microchannels, in each case in a configuration close to a radial arrangement, preferably parallel along both sides of the radial microchannels.

According to a further embodiment not shown, the microchannels 13 of each set comprise all the microchannels set radially relative to the center of the central passage 11a of the substrate 11. That is, the microchannels 13 of each set are slightly angled relative to each other, preferably diverging from each other at the ends further away from the central passage 11, i.e. converging at the ends closer to the central passage 11a.

Each microchannel 13 has an inlet end and is pre-prepared to receive a fluid sample. For this purpose, preferentially, but not necessarily, each microfluidic device M also comprises at least one loading chamber (which may be in the form of a duct or channel) connected by a fluid communication that is the inlet end of each microchannel of the corresponding set 13.

Such loading chambers are clearly displayed in the details shown in Figs. 6 and 7, for example, indicated with 14. From Fig. 7 it can be clearly noted how the microchannels 13 preferably have their inlet ends (some of which are indicated by 13a) in fluid communication with the respective chambers 14, in the case of a parallel connection or arrangement of the ends 13a, or when they are arranged side by side, and how these inlet ends 13a are connected to the chambers 14 themselves. The microchannels 13 thus extend directly from the chambers 14.

In various embodiments, particularly those relating to microfluidic devices provided for centrifugation, the chambers 14 and inlet ends 13a of a given set of microchannels 13 are positioned close to the center of rotation of the substrate 11, and the opposite ends of the microchannels are instead designed to occupy positions further away from the center of rotation.

The microchannels 13 of each set are preferably at least partially identical to one another and/or extend at least partially substantially parallel or equidistant to one another, e.g. substantially parallel or equidistant to one another in the radial direction of the substrate 11. In various embodiments, the microchannels of one and the same set are substantially identical to one another with respect to shape and size. According to other embodiments (not shown), the sets may instead be provided such that the microchannels have substantially one and the same pattern but different lengths from one another.

From FIG. 7 it is noted how, in various preferential embodiments, both the chambers 14 and the microchannels 13 result from cavities or surface etchings made in the substrate 11, the microchannels 13 being in particular in the form of microgrooves. In general, each microchannel 13 may have a width between 5 and 200 μm, preferably between 15 and 50 μm, and/or a depth or height between 2 and 100 μm, preferably between 5 and 40 μm. The length (understood as the distance between its two ends) of each microchannel 13 may be between 5 and 50 mm, as shown. For the sake of analytical uniformity, it is preferred that the microchannels 13 of an identical set have a constant passing section. As shown, the walls or parts of the relief (some of these walls or parts shown by lld in FIG. 7) that separate the microchannels 13 from one another may have a width between 5 and 200 μm, preferably between 15 and 100 μm.

In various preferred embodiments, the chamber 14 has a depth equal to or close to the depth of the microchannel 13, for example, between 2 and 100 μm, preferably between 5 and 40 μm.

As already mentioned, each microfluidic device comprises a cover element 12 at least partially covering the microchannels 13 of the corresponding set of microchannels. The cover element 12 may be made at least in part of a transparent material, e.g. glass or plastic material, to allow observation of the underlying microchannels 13, e.g. for optical detection or illumination purposes. However, this does not constitute an essential feature of the invention, e.g. when the substrate 11 is made of a transparent material, at least in part of which it defines the set of microchannels 13 or in part of which it defines the end regions of the microchannels 13 of a given set.

In various embodiments, such as those described above, one and the same cover element is configured to at least partially cover a set of multiple microchannels 13. For example, referring to the case of Figures 3 and 4, provided on the substrate 11 are 36 sets of microchannels 13, each set side by side or parallel to one another, each having a different length, each substantially radially oriented, and comprising a plurality of microchannel sets all at least partially covered by one and the same cover element 12.

According to other embodiments, each microfluidic device may include one or more individual cover elements, with the or each element at least partially covering a single set of microchannels 13.

The cover element 12 (or each cover element) is configured or sized to leave at least a portion of each microfluidic device M exposed, in particular at least a portion of the chamber 14. To this end, in various embodiments, the cover element 12 has at least one loading opening or passageway that is substantially in the corresponding chamber 14 in the assembled state of the device 10. This feature can be better seen, for example, in Figures 8-10, where some of the loading passageways are indicated at 15. In this example, each loading passageway 15 has a circular profile, although this shape is obviously not required. Likewise, the generally curved profile of the chamber 14 does not constitute an essential feature.

In various embodiments, the material from which the cover elements 12 are made is hydrophilic, facilitating capillary inflow of fluid into each of the set of microchannels 13 from the chambers 14 to the inlet ends 13a of the microchannels themselves. The material from which the microchannels 13 are made, or the material of the substrate 11, may also be hydrophobic in this case.

It is also possible that at least one surface extending over the entire length of the microchannel 13 is made from a hydrophilic material. For example, in a microchannel 13 having a rectangular or trapezoidal cross section, at least one of the four walls defining the cross section of the microchannel is preferably made from a hydrophilic material, for example the wall defined by the cover element 12.

As already mentioned, both the substrate 11 and the cover element 12 may be transparent. For example, the substrate 11 may be made of an at least partially transparent material to allow observation of the microchannel 13, and the cover element 12 may be transparent to allow backlighting of the microchannel itself.

In various embodiments, each microchannel 13 has at least a continuous portion of its inner surface with hydrophilic properties throughout its entire extent. The continuity of the hydrophilic portions along the inner wall of the microchannel 13 can be useful during filling, for example, assuming the deposition of a droplet of sample liquid in the chamber 14 (schematically shown in FIG. 8). Contact with the hydrophilic portions causes filling of the microchannel 13 by capillary action. To this end, in various embodiments, the bottom and side walls of the microchannel 13, as well as the corresponding chamber 14, are made of a single hydrophobic material, while the general parts of the upper wall of the microchannel (for example those parts formed by the cover element 12) are made of a hydrophilic material. On the other side, as can be seen, each microfluidic device is preferentially configured in its end region opposite the inlet end 13a of the microchannel 13 to oppose the exit of the liquid in the absence of stress. As a result, when each microchannel 13 is completely filled, it is no longer subject to the flow of the liquid therein, unless it is subjected to an external force, as will be explained below.

As mentioned above, the substrate 11 of the device 10 does not necessarily have to be disk-shaped. Such a case can be seen from FIG. 8, which shows a microfluidic device M having a substrate 11 having a shape that is substantially parallelepiped, preferably cut in the form of a plane, and a cover element 12 in the form of a foil that is also parallelepiped.

As can be seen, substrates of this kind, i.e. not having a disk shape, can advantageously be pre-arranged for processing, for example via a suitable support or adapter element of a centrifuge device of commercial type, or on a generally disk-shaped support coupled to the rotating member 5a of the device 1 of FIG. 1. It should be noted in any case that FIG. 8 (as well as the subsequent FIGS. 9, 12 and 13) can be understood to represent in each case a portion of a larger microfluidic device, for example the rectangular portion indicated by M of the device 10 of FIG. 3.

The microfluidic device of the device according to the invention comprises, in its end region, substantially opposite the inlet end of the microchannel, a passageway for allowing at least the exit of air from the microchannel itself. According to the invention, provided between this passageway and the microchannel is a filter element permeable to at least air, which is configured to retain particles of interest present in the fluid sample within the microchannel itself.

The mesh or porosity of the filter element can therefore be selected during the manufacture of the microfluidic device according to the size of the particles to be analyzed. In various embodiments, the filter element is also permeable to the liquid portion of the sample fluid, for example to allow the exit of the liquid portion from the microchannel during centrifugation.

In various embodiments, the cover element may also be configured to define at least a portion of a housing sheet for the filter element. Alternatively, the housing sheet for the filter element may be provided within the substrate 11.

In various embodiments, the location of the filter element is generally apical, particularly at a location substantially at the end opposite the end where the fluid enters the microchannel. The microchannel may terminate at a portion of the filter element or extend to a continuous region, further closed by a cover element that is impermeable to both liquids and gases.

In the former case, each microchannel may be completely filled by capillary action, while in the latter case, the region of the channel extending beyond the filter element will initially remain filled with air (unless the filling is performed under vacuum or negative pressure conditions, or the microchannel initially contains at least partially a neutral fluid). During centrifugation, centrifugal forces compress the air (or other fluid), thereby causing its partial or total outflow from the filter element. At the end of the centrifugation, the particles are concentrated at the end of the channel, in the area not covered by the filter element. This configuration is advantageous when the filter element is not transparent or may introduce optical distortions that may worsen the viewing of the pellets, i.e. particle clumps or concentrates. In this way, viewing can be done through a transparent, flat surface.

As mentioned above, a cover element configured to at least partially cover the microchannels of at least one microfluidic device can be sized or configured to define the aforementioned passages. For example, referring to FIGS. 3, 4, 8, 9, and 11, in various embodiments, the cover element 12 defines a passage 16 of a corresponding microfluidic device M, the passage 16 being provided, for example, by a through opening in the element 12. In the case illustrated in FIGS. 3, 4, and 11, if 36 sets of microchannels 13 are provided on the substrate 11, the cover element 12 defines a corresponding number of passages 16. In other embodiments, a single passage 16 can be provided at a position corresponding to multiple sets of microchannels 13.

Again, referring to the example of the figure above, if the microchannels 13 of the various sets are substantially straight, the passages 16 and the loading passages 15 of one and the same microfluidic device are substantially aligned with each other in the extension direction of the corresponding microchannels 13.

3, 4, 8, 9, and 11, designated 17 are at least some of the aforementioned filter elements permeable to air, which should be disposed between the various sets of microchannels 13 and the corresponding passages 16. As can be seen in particular in Figs. 11 and 12, in various embodiments, the cover element 12 may advantageously define a sheet 18 configured for at least partial housing of the corresponding filter element 17. As illustrated (see, for example, Fig. 11), such a sheet 18 may be defined on the side of the cover element 12 facing the corresponding passage 16, in particular the substrate 11.

Thus, in various embodiments, the microfluidic device is configured such that corresponding filter elements are held in an operational position by the same cover element that at least partially covers the corresponding microchannel.

The filter element 17, or each filter element, is preferentially shaped like a membrane, with a porosity or mesh size between 0.02 and 0.45 μm, preferably around 0.2 μm. A preferred class of materials in this sense are ceramic materials, for example alumina, which can be obtained with controlled porosity. In particular, alumina has a very low tendency to bind nonspecifically with the dyes or fluorescent dyes usually used for the marking of cells. It is obviously possible to use other porous materials suitable for the purpose, such as plastic materials, which generally have advantages in terms of cost, but which must be evaluated case by case in relation to their tendency to bind the aforementioned marking dyes or fluorescent dyes, and on the basis of the fluorescence of the polymeric material itself. In general, when the microorganisms or cells to be analyzed have previously been marked with a fluorescent dye, the filter element 17 is preferentially made of a material that does not bind in a nonspecific manner to the fluorescent dye used and does not exhibit autofluorescence, which would cause a decrease in the signal-to-noise ratio.

In various embodiments, the thickness of the filter element used is comprised between 20 and 1000 μm, preferably between 100 and 600 μm. The filter element is preferably optically transparent. Porous alumina tends to scatter light, so it appears opaque and is not suitable as an optical quality substrate, but in certain cases, when its nanopores are filled with water (refractive index of about 1.33) or other fluids with a refractive index close to that of alumina (refractive index of about 1.63 measured at 550 nm), the effects of scattering are greatly reduced and the quality of the images that can be obtained through the wet alumina membrane is sufficient to detect particles or cells in bright field or fluorescence.

As illustrated, element 17 has a rectangular shape, but this shape should not be considered essential. The shape of filter element 17 may vary depending on the actual needs or type of microfluidic device to be obtained.

Figure 8 shows, in a schematic way, a possible introduction mode of a fluid sample into the microfluidic arrangement M of the device according to a possible embodiment of the invention. In the illustrated case, via a suitable tool T (such as a pipette designed to dispense a controlled amount of fluid, representing the order of microliters or tens of microliters), a sample FS of the fluid to be examined is preferably deposited in the chamber 14 through a corresponding loading passage 15 defined at least partially in the cover element 12.

The sample FS may be a simple droplet of fluid, as in the illustrated case, or may have a larger volume.

The chamber 14 and the passageway 15 facilitate the introduction of a fluid sample into the microfluidic device M. Moreover, given that the device M includes multiple microchannels 13, the chamber 14 essentially functions as a collector for the introduction of fluids into several microchannels in parallel. In other words, the provision of a chamber 14 in which the homologous ends 13a of several microchannels 13 are connected in parallel presents the advantage of avoiding the need to introduce each fraction of the sample individually into a single microchannel.

Note that, as previously mentioned, the chamber 14 may be provided by a duct or channel through which the fluid sample is delivered to the inlet end 13a of the microchannel.

The possibility of connecting multiple microchannels to one and the same inlet (whether a chamber, passage or duct) makes it possible to increase the statistical basis of detection, i.e. to utilize multiple repetitions of the same nominal conditions.

The number of microchannels used under the same nominal conditions depends on the type of use of the device and the volume of each microchannel. For example, if the microchannel 13 is 2 cm long, 50 μm wide and 5 μm deep, the total volume will be 5·10 ⁶ μm ^3. A concentration of 10 ⁵ bacteria/mL results in 10 ⁻⁷ bacteria per cubic micrometer. This means that on average there are 0.5 bacteria in each microchannel. This also means that in a microchannel containing at least one bacterium, the signal will double after a very short time (about 20-40 minutes) in case of growth and may remain constant in the absence of growth.

This type of use is sometimes called "digital drug susceptibility." Because the microchannels are very small and can be defined very close to each other in similar patterns, it is possible to have a large number of channels, e.g., 250-500 microchannels, in a very limited area (such as the area of a single microscope slide).

At concentrations such as those just mentioned, it is expedient to allocate a number of 100-200 microchannels to each n-tuplicate (i.e. a set of n microchannels used with the same nominal conditions) at the same nominal concentration in order to obtain sufficient statistical basis. Thus, in a single centrifugable device, for example a disk-shaped device, it is possible to test a large number (various tens) of different conditions, each in n-tuplicates, where n is comprised between 100 and 200. Alternatively, for higher concentrations, it becomes possible to group nominally identical conditions into a small number of microchannels. For example, for concentrations of the order of one million bacteria per milliliter, it becomes possible to use n-tuples of 10-20 microchannels per nominally identical condition.

In various embodiments, each microchannel 13 is closed at its longitudinal end opposite the inlet end 13a, as shown for example in FIG. 15, showing a set of end areas CA of the microchannel 13 where the corresponding filter element 17 is cut off. This kind of embodiment can be used, for example, when a filter element 17 is installed on at least the end stretch of the microchannel 13, as can be seen in FIGS. 13-15. Thus, a fluid can permeate from the inlet end (13a, FIG. 10) of the microchannel 13. This is thanks to the fact that the air contained in the microchannel 13 can be gradually discharged through the filter element 17. It should be noted that the fluid that initially at least partially fills the microchannel 13 can be something other than air (for example a neutral liquid or gas, i.e. without changing the subsequent operation, this initial fluid is replaced by a liquid containing particles that may be of interest for analysis).

From Figures 13 to 15 it can be well noticed how one and the same filter element 17 can be superimposed on several microchannels 13, preferably, but not necessarily, in positions corresponding to their end areas CA, which are parallel to one another and closed at the ends opposite the inlet end 13a.

As mentioned, in various embodiments, each microchannel 13 is preferably filled by capillary action or by taking advantage of the hydrophilicity of at least one of the walls or surfaces that delimit the microchannel itself. On the other hand, as can be seen, in other embodiments (not shown), the fluid sample can be forced into the microchannel under pressure, for example using a positive or overpressure at the inlet or a negative pressure (always with respect to ambient pressure) at the outlet. As mentioned, in the process of filling the microchannel 13, the air originally contained therein can be evacuated through the corresponding filter element 17 and the corresponding passage 16, here defined in the cover element 12.

For example, with reference to the device 10 of FIG. 3, after introducing the corresponding fluid sample into the chamber 14 of at least one apparatus M (for example, as shown in FIG. 8), the microfluidic device is centrifuged, for example, using a device 1 of the same type as that shown in FIGS. 1 and 2. FIG. 16 shows the installation of a disk-shaped type device 10, such as the device of FIG. 3, in a centrifugation and/or detection device 1, with the door 4 of the latter in a closed state.

Following rotation of the device 10 and as a result of the centrifugal forces, particles present in the volume of liquid occupying the microchannels 13 tend to accumulate in their end regions CA and remain primarily within the channel itself. In particular, particles tend to accumulate at or near the closed ends of the corresponding microchannels and/or on their bottom walls and/or on their side walls in the end regions CA near the filter elements 17.

If the filter element 17 is also permeable to the liquid, the same liquid of the sample fluid can pass through the element 17 and the corresponding passage 16 and exit the microchannel 13 as a result of the centrifugal force. However, it will in any case retain the particles of interest in the end region CA of the microchannel.

According to various embodiments, particularly in the case of filter elements 17 that are permeable to liquid, at least a portion of the particles present in the volume of liquid contained in each microchannel 13 tends to accumulate on at least a portion of the filter element 17 located in the end area CA of the respective microchannel 13 or on the portion of the wall of the microchannel bounded by said portion of the filter element 17.

Of course, the dimensions of the microchannel 13 must be sufficient to allow the particles of interest to enter there. In general, relatively shallow microchannels are preferred, i.e. microchannels with a height or depth of the order of the size of the particles of interest, or slightly larger. The reason for this is that, given the same number and size of particles, the amount of particles accumulated alongside one another in the end area CA of the shallow microchannel 13 will form an image in a plane with a larger area than in a deeper microchannel where the particles may overlap one another, thus falsifying to some extent the detection of the amount and/or type of particles. The use of shallow microchannels, preferably with an approximately rectangular cross section, therefore facilitates and improves the quality of the reading of the amount and/or type using an optical system.

For example, if the device 10 has to be used for the separation of different types of whole blood cells, it is preferred that the height (depth) of the microchannel 13 is between 10 and 40 μm, preferably between 10 and 20 μm. Alternatively, if the subject of the analysis is bacteria, the microchannel may have a height (depth) between 3 and 10 μm, preferably between 4 and 8 μm. Again, when measuring yeast, the height (depth) of the microchannel is preferably between 5 and 20 μm, most preferably between 8 and 12 μm.

In any case, thanks to the mentioned device, particles that may be included in the volume of fluid penetrating the microchannel 13 tend to concentrate in the corresponding end area CA as a result of the centrifugal forces directly experienced by the particles, caused by the rotation of the device 10 around the center of rotation 5a, and possibly as a result of the fluid flow and/or the concomitant emptying of the microchannel entraining the particles in suspension.

The detection or reading can be carried out by quantifying by optical methods the size of the particle agglomerates formed in each end accumulation area CA as a result of centrifugation. If the particles have been previously marked with a fluorescent dye, it is also possible to carry out such detection of the amount and/or type by measuring the intensity of the fluorescence.

In various embodiments, the device 1 itself can integrate an optical detection device. The optical device can include a single sensor or an array of sensors (as in the case of an optical scanner, for example) or an array of rectangular sensors, such as CCD or CMOS sensors, capable of capturing an image of the end area CA of the microchannel and analyzing it in various ways, such as an automatic processing program for counting particles. Thus, in general, the same device 1 can integrate the functions of centrifugation and of detection or reading, in particular by utilizing the rotation of the support 10 for both the aforementioned centrifugation for the aforementioned reading using an optical detection device.

16 and 17, for example, illustrate a centrifuge device 1 with a detection arrangement including at least one optical sensor 20, which is itself preferably constituted by an array of optical sensors. In this example, the sensor 20 is in particular stationary and mounted on the bottom wall 3a of the processing chamber 3. The sensor 20 is at a distance from the center of rotation 5a of the device 10, allowing it to pass in front of itself the end areas CA (FIGS. 6 and 15) of all the microfluidic devices present on the disk-shaped device 10. In the illustrated case, the sensor 20 faces the side of the substrate 11 opposite the cover element 12, the substrate 11 being made of a transparent material, at least in the aforementioned end areas CA of the various microfluidic devices M. In this way, the sensor 20 can carry out the necessary optical detection in any case. The optical sensor may be equipped with suitable optics designed to focus and magnify the area of interest.

Possibly, a light source, or other optical detection sensor, can be provided at a portion generally opposite the optical sensor 20 to facilitate optical detection. In the illustrated case, the light source 21 is associated with the inside of the door 3 of the device 1 in a position such that, with the door closed as shown in Figs. 16-17, the light source 21 illuminates at least the end area CA whenever it is exposed to the sensor 20. Also, for this purpose, the filter element 17 may be made of a transparent material or a material that is transparent when in contact with a liquid. A preferred material for providing the filter element 17 is porous alumina, as previously mentioned.

The control system of the device 1 can be preconfigured to control the angular position of the microfluidic device 10 according to the optical detection performed each time. This control system can also be preconfigured to perform the optical detection by driving and stopping the support 10 each time at various angular reading positions after the end of the centrifugation step, or the optical detection can be performed by moving the support 10, preferably at a slow speed, for example by reading slower than the speed during detection or the centrifugation speed, or by synchronizing the rotation with the reading.

In other embodiments, for example with a microfluidic device with microfluidic devices oriented differently than in the case illustrated above with reference to Figures 1 to 3, the optical sensor 20 can be movably mounted on a corresponding guide, for example via its own actuator, so that it can be displaced, for example radially, relative to the device 10 to perform the optical detection required for some microfluidic devices. In such a case, the control system of the device 1 is prepared in advance to control the position of the sensor 21 according to the optical detection performed each time.

In various embodiments of the invention, the optical sensor means 20 of the centrifugation and/or detection device of the type mentioned are configured to acquire an accumulated optical signal or an accumulated image of a plurality of accumulation areas of the microfluidic device, i.e. a signal or an image relating to all accumulation areas CA of the microchannels 13 of the corresponding microfluidic device M. The centrifugation and/or detection device is then pre-prepared, for example via a suitable software, for processing based on said optical signals or images, information representative of the amount of particles accumulated in each of the individual accumulation areas CA of the various microchannels of one and the same microfluidic device, with processing allowing in particular the estimation of the particle number of the individual microchannels 13.

In other embodiments, for example when the optical sensor 20 comprises an array of sensors, such as optical scanners, the sensor itself may be configured to acquire individual optical signals or individual images of the accumulation areas CA of the individual microchannels 13 of the corresponding microfluidic device M. Also in this case, the centrifugation and/or detection device is pre-prepared to process information representative of the amount of particles accumulated in each of the individual accumulation areas CA of the various microchannels of the microfluidic device based on said optical signals or images.

Of course, the device 1 may also be provided to be able to use both of the optical detection techniques mentioned (i.e. collective and individual).

Figures 18-20 show possible variant embodiments of the centrifugation and/or detection device 1 and the microfluidic device 10.

In the illustrated case, the devices 10 have a generally rectangular profile and preferentially each contain a single microfluidic device. However, a device of this shape may also contain several microfluidic devices that are generally parallel to one another.

In particular, as can be seen in FIG. 18, in various embodiments, the device 1 may comprise a centrifuge support 30 defining one or more seats 31 (preferably oriented substantially radially with respect to the center of rotation 5a), each for receiving at least one microfluidic device 10. In various embodiments, the support 30 has in each seat 31 a passage 32 (such as an opening or window or an optically transparent area) that is arranged in a position corresponding to the position assumed by the end detection area CA of the microchannel when the corresponding device 10 is attached to the support itself, as illustrated in FIG. 19. The aforementioned position of the passage 32 on the support corresponds in the radial direction to the position of the sensor 20 of the device 1, so that the sensor can carry out the required optical detection. The passage 32 may be present in the centrifuge support 30 made at least partially of a transparent material, although it makes it possible to make the centrifuge support 30 of an opaque material.

In the non-limiting example shown, four sheets 31 are provided, one for each device 10, with each sheet 31 having a corresponding passageway to enable detection by the optical sensor 20.

In various embodiments, to ensure the positioning of the microfluidic device 10 on the centrifuge support 30, the centrifuge support 30 comprises an upper element, indicated by 40 in FIG. 18, which closes the sheet 31 from above to ensure the maintenance of the position by the microfluidic device 10. The upper element 40 may also comprise a passage 41, such as an opening or window or an optically transparent area, at a position substantially corresponding to an end region of the microfluidic device of the device 10, to allow its illumination by the light source 21.

Figure 20 shows diagrammatically the mounting of a support 30 with corresponding top elements 40 between which a microfluidic device 10 is set, only one of which is visible in the cross-sectional portion of the top element 40. The operation of the device 1 of Figures 18 to 20 is similar to that of the device 1 described with reference to Figures 1 to 2 and Figures 16 to 17 with regard to the centrifugation and/or detection functions.

Figure 21 shows a microfluidic device 10 with a rectangular profile, suitable for use in a centrifugation and/or detection device 1 of the type shown in Figures 18 to 20, i.e. designed to be mounted on a corresponding centrifugation support 30. In this case, the device comprises a single microfluidic device M, which in turn comprises a set of microchannels 13 defined on a substrate 11, as well as a chamber 14, a cover element 12 and a filter element 17, as can be seen from Figures 22 or 23.

22, the filter element 17, having a substantially rectangular profile, is sized to completely or substantially completely coat the microchannel 13 and leave at least a portion of the chamber 14 exposed. The material of which the filter element 17 is made may advantageously be a hydrophilic or hydrophobic material, as required, based on what has been explained above. The filter element 17 may be fixed in place on the substrate 11, for example via gluing or bonding. Then, on the filter element 17, and possibly partially on the substrate 11, a cover element 12, also having a substantially rectangular profile, is fixed.

23, the filter element 17, having a substantially rectangular profile, is sized to coat only the end region of the microchannel 13 opposite the chamber 14. Also in this case, the filter element 17 can be fixed in place on the substrate 11, for example via gluing or bonding. Then, at least partly on the filter element 17, possibly partially on the substrate 11, a cover element 12 is fixed, again having a substantially rectangular profile, in this case directly delimiting the microchannel 13 at its top, at least for its substantial stretch extending between the filter element 17 and the chamber 14.

22 and 23, the element 12 is sized in such a way that a passage 16 is defined for the outflow of air and possibly liquid of the fluid sample, in any case according to what has already been described before, and in each case at least a part of the chamber 14, as well as at least one end of the filter element 17, remains exposed.

22 and 23, the cover element 12 extends at least partially over the microchannel 13, but has a filter element 17 set at least partially between the microchannel 13 and the cover element 12. The element 12 is substantially impermeable to the fluid, so that in this way it is possible to confine the fluid itself within the microchannels 13, at least between their inlet ends 13a (i.e. chambers 14) and their accumulation portion CA, where the filter element 17 is not covered by the cover element 12. However, with reference to the embodiment of the type shown in FIG. 23, it is noted that the cover element 12 does not necessarily have to be at least partially superimposed on the filter element 17, but rather that these two elements can be fixed in adjacent positions on the substrate.

Figures 24-25 illustrate possible modes of introducing a fluid sample FS into the microfluidic device according to Figures 21-22, using a suitable tool T, as already explained with reference to Figure 8. From Figure 24, one can particularly see the inlet end 13a of the microchannel 13, which, as in the illustrated case, can be covered on top by a filter element 17. From the following Figure 26, one can see instead the opposite end of the microchannel 13, closed at their longitudinal ends, in order to define an end area CA of accumulation of particles after centrifugation.

As can be seen, again in this case the concentration of the particles of interest at the end region CA of the microchannel 13 is obtained by rotating the device 10 about a center of rotation, for example using a device 1 of the type shown in Figures 18-20.

As already mentioned, in various embodiments, the microchannel 13 can extend beyond the filter element 17 in an area that is closed by the cover element 12 anyway. One such case is illustrated in FIG. 26a, where an end area CA of the microchannel 13 is highlighted, which extends beyond the filter element 17 but is anyway covered by the cover element 12 with the passage 16. In this case, the filter element 17 and the passage 16 are therefore in the intermediate area of the microchannel, i.e. upstream of the corresponding closed end area CA. The area CA is initially filled with air (or other gas or neutral liquid), which can be compressed during centrifugation and exit partially or completely through the filter element 17 and the passage 16. At the end of the centrifugation, the particles are concentrated at the lower end of the microchannel 13, i.e. in the area CA that is not covered by the filter element 17. In this type of embodiment, the filter element is therefore not transparent, and at least one of the substrate 11 and the cover element 12 can be transparent, in order to allow the necessary detection.

As mentioned above, in other embodiments, the fluid sample can be forced under pressure through the microchannels of the microfluidic device according to the invention, for example using overpressure at the inlet or negative pressure at the outlet relative to ambient pressure, thus even in the absence of centrifugation. Examples of this kind are shown in Figs. 27 and 28 in relation to device 10 as in Fig. 21.

27, a pressure generator system is provided for this purpose, which is only partially visible and is indicated by 60, and which is prepared in advance to generate a pressurized flow of the liquid to be treated, or of air or other gas A, and to direct it into the chamber 14 in which the sample of the fluid has previously been set up. In this way, the fluid sample in the chamber 14 is forced to first penetrate the microchannels 13, then pass them to their closed ends and then exit from the passage 16, possibly through a corresponding part of the filter element 17, in which case it also penetrates the liquid. In this way, the pressurized gas or fluid brings about the exit of the liquid fraction of the sample from the microchannel 13, in the end region of which, according to what has been described above, the particles that may be to be analyzed are instead accumulated.

Instead, FIG. 28 illustrates a system for generating negative pressure or vacuum, only partially visible and indicated at 70, such as a suction syringe or pump, pre-arranged to generate a vacuum or suction pressure V at the passage 16 defined by the end stretch of the filter element 17, in this case permeable to liquid.

In this way, the fluid sample previously set in the chamber 14 is drawn in thanks to the generated negative pressure or vacuum, first penetrating the microchannels 13, then passing them to their closed ends and finally exiting, again from the passage 16 through the corresponding part of the filter element 17. In this way, the end regions of the microchannels are instead filled with possible particles to be analyzed, while the liquid part is expelled from the device 10.

It will be appreciated that the pressure generator system 60 and/or the suction system 70 may also be used in the case of a microfluidic device 10 of the type shown in FIG. 3.

In various embodiments, the microchannels of the microfluidic device M are used solely for the detection of particles of interest contained in the fluid sample, but in other embodiments, the microchannels can also be utilized as culture wells, particularly if the particles to be detected are microorganisms capable of reproduction. Alternatively, some microchannels can be "loaded" with biological material (e.g., bacteria) that have been induced to grow outside the device or inhibited by antibiotics.

In various embodiments, at least one or each microchannel may be associated with at least two electrodes, in particular at least in the respective end areas CA. These electrodes may be electrodes for detection and/or electrodes for manipulation of the particles.

For example, in various embodiments, at least one pair of electrodes in the end area CA can be used to perform a reading of the amount of particles via detection of electrical impedance. It is also possible to perform a differential reading by arranging a further pair of electrodes in the part of the microchannel comprised between the corresponding ends, in order to make it possible to distinguish the contribution to the electrical impedance represented by the particles from that represented by the sample fluid. If the liquid sample is a culture medium or a physiological solution, the electrical conductivity is relatively high due to the ions dissolved in the liquid.

Pairs of electrodes arranged such that one electrode of the pair is at a position corresponding to the part of the microchannel close to the corresponding inlet end 13a and the other electrode of the pair is near the end region also allow verification that the microchannel is properly filled with the fluid containing the particles to be counted (this verification is relatively easy, given that the fluid has a much higher electrical conductivity than air, which is generally an insulator). Preferably, the electrodes, if envisaged, are also made at least partially from a conductive transparent material.

Given that the device 10 of the present invention can be used to accumulate cells at a precise location (i.e., at the end accumulation area CA), electrodes of the type mentioned can also be used to perform operations on the cells themselves, e.g., electroporation, or to maintain them in place by dielectrophoresis.

As already mentioned, the microfluidic device 10 according to the present invention can be used for the purpose of simple counting and/or detection of the types of particles contained in a fluid sample, or also to perform more complex functions of analysis, such as an antibiogram (in this case the microchannels can also be pre-treated, for example by introducing an antibiotic).

The microfluidic device and the centrifugation and/or detection device according to the invention can be advantageously used for the purpose of evaluating the growth capacity of bacteria and pathogens and, subordinately, for the purpose of determining their antibiotic susceptibility profile (antibiogram) in a short time and with a small volume of sample liquid.

Methodologies known for this purpose are based on the assessment of the ability of a pathogen or bacterium to form colonies in a medium suitable for its growth, or on the assessment of the turbidity of the culture broth after the growth of the pathogen. The ability of an antibiotic to inhibit the growth of a pathogen or bacterium is classically assessed by counting the corresponding colonies or on the level of turbidity of the corresponding culture broth, a property that varies as a function of the sensitivity of the pathogen or bacterium to the antibiotic.

This sensitivity is related to the ability of the antibiotic to inhibit the efficient growth of the bacterial strain, and it is clear that the time associated with this type of analysis depends on the rate at which the pathogen or bacteria grows. The approach carried out according to conventional techniques is essentially based on the fact that a "two-dimensional" layer (colony) of bacteria or pathogens can grow until it becomes visible to the naked eye, or that the growth of bacteria or pathogens in a liquid can be such that it modifies, in a statistically significant way, the turbidity of the liquid itself, which can be measured by photometry in the turbidity range (readings are usually taken at wavelengths between 500 and 600 nm).

The technique proposed here, making use of the aforementioned microfluidic device, is instead based on several parameters that do not take into account either the two-dimensional growth of the layer of bacteria or pathogens or their growth in the liquid, which is read as an increase in turbidity.

More specifically, the methodology proposed in this specification envisions the following:

i) obtaining short-term growth of biological material (e.g. urine collected directly by a patient) with or without the addition of growth factors (e.g. bacterial culture broth such as BH);

ii) introducing the culture obtained in the previous step into a microchannel 13 seeded with the same concentration of biological material and/or culture medium (for this purpose it may be particularly advantageous to provide a microwell in the microchannel 13);

iii) measuring the growth of bacteria in the microchannel 13;

iv) identifying one or more "negative" microchannels 13, i.e., those to which only culture medium is added (e.g., 50% with PBS buffer or physiological solution);

v) identifying one or more "positive" microchannels 13, i.e. those capable of verifying the growth capability of bacteria or strains present in the system of microchannels 13;

vi) identifying a series of microchannels 13 containing antibiotics in such a way as to verify the resistance or susceptibility of bacteria or strains present in the seeded biological material to the antibiotics;

The measurement of susceptibility to antibiotics can be carried out using different strategies starting from the deposition of the bacteria or pathogen after growth in the accumulation area CA of the microchannel 13. This can be obtained via centrifugation of the device 10 or via overpressure and/or negative pressure as described in relation to Figures 26-27. This approach advantageously makes it possible to carry out the necessary comparisons between:

- the amount of bacteria or pathogens present in the starting material;

- the amount of bacteria or pathogens present at the end of incubation;

- The amount of bacteria or pathogens present in the antibiotic-treated microchannel 13; the use of appropriate fluorescent dyes may allow selective discrimination between live and dead bacteria.

Particularly advantageous for this type of analysis can be a support 10 equipped with several microfluidic devices, such as the support of FIG. 3. These microfluidic techniques have a higher sensitivity compared to other techniques (such as turbidity), since they allow to "concentrate" the microorganisms in a small space by external stress (centrifugation, overpressure, negative pressure), thus visualizing them in bright field with visible light, both in transmission and reflection, or with the fluorescence of marked cells. With the proposed concentration technique and a suitable analysis of the images, either with a linear array of sensors or with a rectangular array of sensors (for example with a CCD or CMOS camera or other technology used for image acquisition), a change of +/- 20% in the number of cells is measured in a reliable and precise manner. This degree of variation, which can be detected using the proposed methodology and which cannot be detected instead using classical turbidity techniques, can be determined even after short growth times, for example between 20 and 40 minutes. The quantification or estimation can be carried out via optical detection, as mentioned above, at least in the accumulation area CA of the various microchannels 13 of interest.

Additionally or alternatively, a count of bacterial bodies can be performed using electrodes set in the accumulation area CA to detect a change in impedance of the electric field containing a "growing" population of bacteria or pathogens. This change can be used as a signal of the susceptibility (or resistance) of the bacterial strain under test. Again, detection times can be extremely short.

The above methodology can be beneficially used in extremely different clinical situations.

For example, it is possible to measure the "absolute" number of bacteria or pathogens in a sample of relatively common biological material (e.g., urine for urine culture). Where a count of, for example, more than 100,000 bacteria/mL indicates an infection of the urinary tract, mere "numerical" documentation of the bacterial charge is quite accurate in indicating the pathological situation.

Also, in the absence of bacterial or pathogen identification (which can be performed with standard techniques if necessary), the susceptibility/resistance profile to an antibiotic panel can be easily assessed, offering the patient the opportunity to receive a "non-empirical" treatment, but based on a real antibiotic susceptibility study. In this case, it is important to recall that the majority of positive urine cultures are characterized by a single isolated pathogen, whereas polymicrobial infections occur more frequently in hospitalized patients, or in patients complicated by preanalytical causes and for reasons related to the sampling technique.

In more complex situations (e.g. hospitalized patients), the identification of bacteria leads to improved treatment strategies for the patient as well as for hospital-acquired infections that may be associated with it. On the other hand, as already mentioned, in the case of non-"noble" materials such as urine, the identification of the pathogen can follow various routes, but if the antibiotic susceptibility profile is not performed in a very short time, the setting of a life-saving antibiotic therapy may be delayed. For this reason, the device 10 (particularly with microwells, as already mentioned) can be loaded with single colonies (e.g. isolated from blood cultures) that have not yet been identified but that require an immediate therapeutic approach. In this latter case, bacteria isolated from complex material can be seeded and an antibiogram can be available within a few tens of minutes.

From the above description, the features of the present invention are clear, as are its advantages.

The proposed device and methodology allow working with relatively small starting sample volumes, for example 0.05-1 mL. For example in pediatrics, in studies carried out on small animals and where it is useful to reduce the amount of (biological and reagent) material, it is advantageous to be able to use relatively small volumes, also for economic reasons. The measurement of the particulate component ends once the number of particles has been counted, such that reproducibility problems are virtually nonexistent. Typically, 16,000 particles are counted to obtain an accurate estimate of a subpopulation represented by 1-5% of the total. Thus, assuming, for example, starting from a concentration of 100,000 particles per milliliter, according to the invention a starting sample volume comprised between 0.2 and 0.4 mL is sufficient, while for higher concentrations, for example 1 million particles per milliliter, the starting sample volume can drop to, for example, 0.02-0.06 mL.

The device according to the invention is particularly advantageous for carrying out antibiograms.

Generally speaking, for this purpose, a bacterial culture may be inoculated into the microchannel 13 of at least one unit M of the device 10. The device 10 is then subjected to centrifugation, or overpressure, or underpressure, as described above, and the number of bacteria accumulated in the area CA of the microchannel 13 is then quantified or estimated. In this type of application, the microfluidic device 10 may be used exclusively for the quantification of microorganisms, e.g. bacteria, as long as the growth in the different conditions to be compared can previously be obtained using conventional laboratory equipment and devices.

In other applications, the antibiogram can be carried out starting from a two-dimensional culture of bacteria on a solid support. In this case, the methodology can envisage the following steps:

i) harvesting bacterial colonies from solid culture plates;

ii) inoculating a colony or a portion thereof into a liquid medium, e.g., a culture broth, preferably to form a homogenous dispersion;

iii) loading the liquid medium containing the bacteria into the microchannels 13 of at least one of the units M of the device 10, including at least some of the aforementioned microchannels previously provided with an antibiotic, preferably a lyophilized antibiotic of a different type and/or concentration, and other microchannels in which no antibiotic has been provided;

iv) incubating for a period ranging from 10 minutes to 6 hours, preferably 1 to 2 hours;

v) processing the device 10 via centrifugation, or overpressure, or underpressure;

vi) quantifying the bacteria accumulated in the area CA of the microchannel 13, in particular by performing a relative quantification between the microchannel 13 pretreated with the antibiotic and the microchannel 13 that has not been pretreated, in order to obtain a profile of the sensitivity of the bacteria in question to the antibiotic(s) used.

In yet another application, the device according to the invention can be advantageously used to carry out an antibiogram starting from a primary sample, i.e. a sample taken directly from a subject or host organism (human or animal). In this case, the methodology can envisage the following steps:

i) Obtaining a bacterial concentrate or mass (or pellet) from the primary sample, e.g. urine; for this purpose, for example, the primary sample may be centrifuged using conventional laboratory equipment and devices to separate said bacterial mass from the surfactant. Centrifugation is preferably carried out in two steps: a first step at low speed to eliminate the cells; a second step at high speed to concentrate the bacteria; alternatively, the first centrifugation step can be replaced by filtration to eliminate the cells.

ii) inoculating the resulting bacterial mass or a portion thereof into a liquid medium, e.g., a culture broth, preferably to form a homogeneous dispersion;

iii) loading the liquid medium containing the bacteria into the microchannels 13 of at least one device M of the support 10, including at least some of the aforementioned microchannels previously provided with an antibiotic, preferably a freeze-dried antibiotic of a different type and/or concentration, and other microchannels in which no antibiotic has been provided;

iv) incubating for a period ranging from 10 minutes to 6 hours, preferably 1 to 2 hours;

v) processing the device 10 via centrifugation, or overpressure, or underpressure;

vi) quantifying the bacteria accumulated in the end region CA of the microchannel 13, in particular by performing a relative quantification between the microchannel 13 pretreated with the antibiotic and the microchannel 13 that has not been pretreated, in order to obtain a profile of the sensitivity of the bacteria in question to the antibiotic(s) used.

It will be apparent to those skilled in the art that numerous modifications can be made to the supports and substrates, devices, and methods described herein by way of example without departing from the scope of the present invention. Similarly, it will be apparent to those skilled in the art that individual characteristics described in connection with one embodiment may be used in other embodiments described herein, even if they differ from the previous example.

The application of the present invention is not limited to the medical or veterinary sector, but it is possible to use the described supports and devices for the concentration and/or quantification of particles present in any type of fluid, for example also in the industrial or agricultural fields.

Claims (15)

A microfluidic device for concentrating particles contained in a fluid sample (FS), comprising a substrate (11) having a surface (11b) on which at least one microfluidic device (M) is defined, said microfluidic device comprising:
a loading chamber (14) for loading a fluid sample (FS) into at least one microfluidic device (M);
a plurality of microchannels (13) having respective inlet ends (13a) extending from a loading chamber (14), the plurality of microchannels (13) being fluidly connected in parallel to said chamber (14);
a cover element (12) that is substantially impermeable to the fluid sample (FS) and that extends at least partially across the plurality of microchannels (13),
the loading chamber (14) and the plurality of microchannels (13) extend substantially according to a plane defined by a substrate (11);
- said loading chamber (14) and said plurality of microchannels (13) are in the form of cavities or surface etches of a substrate (11);
- each of said plurality of microchannels (13) is closed at its longitudinal end opposite the inlet end (13a);
- said plurality of microchannels (13) are partially delimited, at least in their accumulation areas (CA) substantially opposite their respective inlet ends (13a), by a filter element (17) that is at least permeable to air and that is located over at least a portion of said plurality of microchannels (13) and that is configured to retain particles that may be present in a fluid sample (FS) within each of said plurality of microchannels (13);
a microfluidic device in such a way that particles that may be contained in the volume of fluid of the fluid sample (FS) penetrating each microchannel (13) tend to concentrate in the respective accumulation area (CA) as a result of forces applied to at least one of the microfluidic device (10) and the fluid sample (FS) loaded in the loading chamber (14), including centrifugal forces caused by rotation of the substrate (11) about the center of rotation (5a), or positive pressure applied to the fluid sample (FS) in the loading chamber (14), or negative pressure applied to the fluid sample (FS) in the accumulation area (CA) of the microchannel (13). The cover element (12) comprises:
a passage (15) for introducing a fluid sample (FS) into the loading chamber (14);
- a passage (16) for exhausting air from the plurality of microchannels (13) through a filter element (17);
- the passage (16) for draining the liquid from the plurality of microchannels (13) and the filter element (17) are permeable to the liquid;
The microfluidic device of claim 1 , which is sized or shaped to define at least one of: The microfluidic device of claim 1, wherein at least one of the substrate (11) and the cover element (12) is configured to define at least a portion of a seat (18) for a filter element (17), and/or the cover element (12) is configured to maintain the filter element (17) in a corresponding operating position. The microfluidic device according to any one of claims 1 to 3, in which only the terminal ends of the multiple microchannels are set upward. Each of the plurality of microchannels (13) comprises:
- has a width of 5 to 200 μm, and/or - has a depth or height of 2 to 100 μm, and/or - has a length of 5 to 50 mm, and/or - has a substantially constant passage section,
A microfluidic device according to any one of claims 1 to 4. - each of the plurality of microchannels (13) has at least one surface portion defined by at least one of a hydrophilic material and a hydrophobic material, said hydrophilic material and/or said hydrophobic material belonging to at least one of the substrate (11), the cover element (12) and the filter element (17), and/or - said device (10) is at least partially made from a transparent material, said transparent material belonging to at least one of the substrate (11), the cover element (12) and the filter element (17),
A microfluidic device according to any one of claims 1 to 5. The substrate (11) is
- configured for attachment to a rotating member (5a) of a centrifugation and/or detection device (1), in particular via an adapter support (30, 40), and/or - substantially disc-shaped,
A microfluidic device according to any one of claims 1 to 6. At said surface (11b) of the substrate (11), a plurality of said microfluidic devices ( M ) are defined; and/or a plurality of microchannels (13) are set side by side or extend at least in part substantially parallel or equidistant to one another or comprise a first microchannel (13) set radially relative to the centre of rotation (11a) and a second microchannel (13) set parallel or equidistant to said first microchannel (13); and/or said filter element (17) comprises a membrane having a porosity or mesh size between 0.02 and 0.45 μm,
A microfluidic device according to any one of claims 1 to 7. the membrane is at least partially made of a film of ceramic material, in particular a film of porous alumina;
The microfluidic device of claim 8. A system for concentrating particles contained in a fluid sample (FS), comprising a centrifugation device and a microfluidic device according to any one of claims 1 to 9, the centrifugation device having a rotating member (5a) configured to rotate the microfluidic device (10). A system for detecting particles contained in a fluid sample (FS), comprising a detection device (1) and a microfluidic device (10) according to any one of claims 1 to 9, said detection device (1) having a rotating member (5a) configured to impart an angular motion to said microfluidic device (10) and optical sensor means (20) configured to perform optical detection on the microfluidic device (10) and/or to detect particles accumulated in an accumulation area (CA) of the microfluidic device (10),
said optical sensor means (20) being configured to acquire optical signals or images of one or more accumulation areas (CA) of the microfluidic device (10);
said detection device (1) being pre-prepared for processing information representative of the amount of particles accumulated in an accumulation area or areas (CA) based on said optical signal or image,
system. 1. A method for detecting particles possibly present in a fluid sample (FS), comprising:
a) providing a microfluidic device (10) according to any one of claims 1 to 9;
b) introducing a volume of a fluid sample (FS) into a loading chamber (14) of at least one microfluidic device (M) of the microfluidic device (10);
c) centrifuging the microfluidic device (10) or subjecting the corresponding fluid sample (FS) to positive or negative pressure in the loading chamber (14) or accumulation area (CA) of the microchannel (13), respectively;
d) detecting, in particular by optical and/or electrical methods, particles that may have accumulated in the accumulation area (CA) of each microchannel (13);
The method includes: - step b) comprises providing a liquid medium containing a microorganism, a pathogen or a bacterium of at least one bacterial strain,
- step d) comprises quantifying the number of microorganisms, pathogens or bacteria accumulated in the accumulation area (CA) of each microchannel (13);
The method of claim 12. i) pre-treating a first microchannel (13) with at least one first antibiotic;
ii) obtaining a mass of microorganisms, pathogens or bacteria;
iii) inoculating at least a portion of said mass into a liquid medium;
iv) introducing a quantity of a liquid medium into said first microchannel (13);
iv) waiting for a period of between 10 minutes and 6 hours;
v) centrifuging the microfluidic device (10) or subjecting the corresponding fluid sample (FS) to positive or negative pressure in the loading chamber (14) or accumulation area (CA) of the microchannel (13), respectively;
vi) quantifying the number of microorganisms, pathogens or bacteria accumulated in the accumulation area (CA) of said first microchannel (13), in particular by performing a relative quantification between said first microchannel (13) and a second microchannel (13) of the microfluidic device (10) that has not been pretreated with the at least one first antibiotic, in order to obtain a susceptibility profile of said microorganisms, pathogens or bacteria to the at least one first antibiotic;
The method of claim 13, comprising the steps of: The method according to claim 14, wherein the mass of operation ii) is obtained from a sample taken from a culture dish or from a host organism, in particular by centrifugation or by filtration and centrifugation of the sample.

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